Overview of Research Projects

Cellular Pathways in Bone Mineralization

Judith Schaart

Supervisors: Anat Akiva, Nico Sommerdijk

Bone consists of a mineralized and highly organized collagen matrix that is produced and maintained by multiple cell types, including osteoblasts. During bone development osteoblasts secrete a collagen matrix which is mineralized with hydroxyapatite. The pathways underlying bone mineralization have not been completely elucidated yet. Most in vitro studies into bone have been performed in 2D or in static 3D cell culture systems, however, in vivo bone development is also influenced by mechanical stimuli, for instance caused by walking.

We aim to develop a bone-on-a-chip to create a flow-controlled 3D culture on which mechanical stimulation can be performed. With this device, we will mimic the in vivo conditions for osteogenesis more closely and improve the organization of the collagen deposited by osteoblasts. After establishing this culture system, bone mineralization pathways will be studied using a combination of light and (cryo-) electron microscopy.

Pascal Jonkheijm, Andries van der Meer & Dorothee Wasserberg (University of Twente)
Willi Jahnen-Dechent (RWTH Aachen)

Osteogenesis Imperfecta

Robin van der Meijden

Supervisors: Anat Akiva, Nico Sommerdijk

Osteogenesis imperfecta (OI) is a genetic disease that alters the structure of the collagen. This change in collagen structure results in weakening of the patients' bones. However, the way this change in collagen structure affects the bones so strongly, and why there is a large difference between patients remains largely unknown.

We aim to gain more insight into both the changes in chemistry and structure of the bone tissue from patients suffering from OI. We will analyze bone tissue from patients with osteogenesis imperfecta using cryo-scanning electron microscopy (cryo-SEM) and Raman microspectroscopy. Cryo-SEM allows high resolution imaging of the structure of bone. Information about the chemical environment of the collagen matrix as well as the hydroxyapatite minerals can be obtained with Ramen microspectroscopy. This combination of chemical and structural information will give new insights in the progress of the disease.

Harrie Weinans, Wouter Nijhuis & Ralph Sakkers (UMC Utrecht)

The Role of Collagen Associated Carbohydrates in Bone Biomineralization

Luco Rutten

Supervisors: Elena Macías Sánchez, Nico Sommerdijk

Bone is a hierarchical structure which is essential for mechanical support and protection, consisting of a collagen matrix mineralized with carbonated hydroxyapatite. During the formation of the collagen matrix undergo post-translational modifications such as intracellular glycosylation of hydroxylysine residues. Diseases which have been related to weaker bones, such as osteogenesis imperfecta and diabetes, show an increased carbohydrate content. However, the role of collagen glycosylation in the mineralization process is not known.

In this project we will establish different collagen model systems with varying degree of glycosylation to investigate the effect on the mineralization process. This includes the mineralization in a single fibril were the parameters are well controlled and the mineralization of tissue were the mineralization environment is still intact. Investigating these model systems liquid phase electron microscopy, we aim to observe the mineralization at the nanoscale in real time and investigate the influence of carbohydrates during the mineralization of bone.

Ruud Bank (UMC Groningen/University of Groningen)

The Role of Citrate in Bone Mineralization

Chenglong Li

Supervisor: Nico Sommerdijk

Bone mineral (apatite) is hierarchically assembled in a collagen matrix, from the nanoscale to the multi-micron level: featuring needle-shaped crystals, platelets, stacks of roughly parallel platelets and eventually mineral aggregates in continuous, cross-fibrillar mineralization. Recent high-resolution imaging has revealed that many of the bone apatite crystals show strong curvature. Where collagen is thought to determine the shape and orientation of the intrafibrillar crystals, a key, unanswered question is what determines the mineral shape and orientation in the extrafibrillar area.

Since it was demonstrated that ~90% of the bulk citrate of the human body resides in mineralized tissues, the relationship between citrate and the bone has been intensely discussed. Here, we explore how citrate may play a role in controlling bone mineral development. For this we use cryo-TEM and Raman to obtain in-situ chemical, structural and morphological information on the role of citrate.

Melinda Duer (University of Cambridge)

Funded by:
China Scholarship Council (CSC) 

Matrix Vesicles and Collagen Mineralization

Marcos Eufrásio Cruz

Supervisors: Anat Akiva, Nico Sommerdijk

Biomineralization of hard tissues involves a complex spatio-temporal sequence of events regulated by bone-forming cells. One big question that still perdures on the understanding of this process is the origin of the mineral phase. It has been claimed since their discovery in the 1960s, that matrix vesicles (MVs) could be responsible to nucleate and deliver calcium phosphate to mediate the ECM calcification. However, besides more than 50 years since their first observation, the true nature and function of MVs are still under debate and the molecular pathways behind their calcification are just beginning to be uncovered.

Our aim is to understand how MVs are filled up with calcium phosphate in the extracellular space and how these mineral-filled vesicles can mediate collagen mineralization. To visualize the MVs in situ and to track them from their formation up to their interactions within the ECM, we will correlate the information from 3D light microscopy with that from 3D high-resolution electron microscopy (3D FIB/SEM).


Ana Paula Ramos (University of São Paulo)

Funded by:
FAPESP - São Paulo Research Foundation

CLEM Workflow for NCP Visualization

Marit de Beer

Supervisors: Anat Akiva, Nico Sommerdijk

Non-collagenous proteins (NCPs) are involved in guiding the mineralization of the extracellular bone matrix. To understand their roles in the bone formation process we set out to visualize these NCPs "at work", i.e. when and where they perform their action in the extracellular matrix. 

For this we will apply CRISPR-Cas9 technology to create fluorescently labeled proteins that will appear during osteoblast differentiation in a human cell culture system. To place the time resolved localization of the NCPs in the ultrastructural context, we will correlate the information from 3D light microscopy with that from 3D high resolution electron microscopy (3D FIB/SEM). To obtain the most reliable information we will use a combination of live imaging with cryo-florescence and cryo electron microscopy via direct high pressure freezing.